An ever-expanding family of ets-related genes has been found in the human genome, as well as in that of other species. Features of this ets family include a DNA-binding domain, as well as a helix-loop-helix motif, which has been postulated to be a site for protein-protein interaction. The goal of this project is to identify proteins which interact with individual ETS proteins, and thus give rise to functional differences among the members of the ets family. We have demonstrated the existence of interacting proteins using protein blotting techniques, and have been utilizing the yeast two-hybrid system to find the genes encoding these ets-interacting proteins. ETS1, ETS2, and ERGB/FLI1 genes have been placed into shuttle vectors which express the Ets proteins fused with the yeast GAL4 DNA-binding domain. Using this system, we have been able to assess the relative transcriptional activation levels of these ETS family members, as well as those of the alternatively-spliced forms of ETS1. Using a cDNA library constructed from T-lymphocyte CEM cells, we have obtained several clones which interact with ETS1 or ERGB/Fli-1. Unfortunately, none of these clones demonstrated interaction when tested in a non- yeast-based system, suggesting that yeast proteins may have bridged the ETS/library interaction. Alternatively, these clones could represent false-positives commonly found in the two-hybrid system. In an effort to decrease the number of false-positives obtained in the yeast two-hybrid system, we have started to develop a yeast 1.5 hybrid system which does not rely on GAL4 interaction with a GAL4-regulated promoter to activate the reporter gene. Instead, this 1.5 hybrid system uses the DNA-binding domain of the ETS family member itself to interaact with a consensus ETS-binding DNA sequence linked to the reporter gene. Several such reporter constructs have been established and this new system is being tested.